Migraine affects predominantly females in their fertile years, and in close to half of all women migraineurs, the frequency and severity of migraine attacks increase around the time of menstruation. Although the mechanisms underlying these menstrually-related migraines are unclear, the abrupt decline in plasma estrogen levels during the premenstrual phase is now believed to be a critical factor. How estrogen withdrawal promotes menstrual migraine remains nevertheless speculative. Based on preliminary experiments in female rats, we hypothesized that changes in the level of reproductive hormones exert a modulatory influence on the activity of meningeal nociceptors and specifically, that estrogen withdrawal produces an increase in meningeal nociceptor sensitivity to migraine triggers such as glyceryl trinitrate (GTN). We further hypothesized that this enhancement in meningeal nociceptor activity subsequently facilitates the development of delayed pericranial hypersensitivity (a condition associated with migraine pain). Finally, we postulated that one possible mechanism underlying this pro-nociceptive effect of estrogen withdrawal may be related to the influence it exerts on dural MCs, which in turn can promote meningeal nociceptor activation. We propose to test our working hypotheses using in-vivo electrophysiological recordings as well as behavioral and histological techniques. Three specific aims are proposed: Aim 1 is to determine whether estrogen withdrawal, either occurring naturally during the estrous cycle or induced experimentally in ovariectomized (OVX) animals modulates the responses of meningeal nociceptors to infusion of the migraine-triggering molecule GTN. Aim 2 is to examine whether the enhancing effect of estrogen withdrawal on GTN-evoked activation of meningeal nociceptors is manifested behaviorally as pericranial tactile hypersensitivity. Aim 3a is to examine the relative contribution of MCs in mediating the effect of estrogen withdrawal on GTN-evoked meningeal nociceptor activation and pericranial hypersensitivity using treatments that either deplete MCs or block their degranulation. Aim 3b is to explore the possibility that one mechanism by which estrogen withdrawal enhances the effect of GTN is by affecting the propensity of dural MCs to become degranulated in response to GTN. Taken together these novel studies seek to advance the understanding of the pathophysiology of migraine and in particular, mechanisms that may play a role in promoting migraine attacks associated with the menstrual cycle in females. PUBLIC HEALTH RELEVANCE: Migraine is three times more prevalent in women, and in about half of all women migraineurs, headache frequency and severity increase around the time of menstruation, a hormonal phase associated with estrogen withdrawal. The project will study a potential mechanism underlying menstrual migraine by examining the effect of estrogen withdrawal on neuronal and behavioral correlates of migraine pain in rats. The study will further examine the possibility that one mechanism underlying the effect of estrogen withdrawal may be related to its influence on dural mast cells.